Abstract How transcriptional asymmetry arises in the epiblast before gastrulation remains a central question in mammalian development. Using a targeted CRISPRa screen, we identify Wt1 as a regulator of lineage-associated transcriptional programmes during formative pluripotency. Wt1 is transiently induced during formative pluripotency in vitro and in vivo, coincident with emerging lineage biases. Precocious Wt1 induction advances cells toward post-implantation identity, whereas Wt1 -deficient cells execute naïve exit but show impaired acquisition of formative and lineage-associated programmes. WT1 binds active regulatory elements together with OTX2, OCT4 and FOXO1. Comparison of Wt1 splice isoforms identifies KTS splice status as the major determinant of divergent anterior/neuroectodermal and posterior/mesodermal transcriptional programmes in vitro. In the E5.5 epiblast, Wt1 expression and splice composition align with lineage-biased states, linking isoform usage to anterior bias in vivo. Isoform-dependent programmes are conserved in human pluripotent cells. These findings identify alternative splicing as a mechanism tuning lineage-associated transcriptional bias before gastrulation.